Role of Runt-related Transcription Factor 3 (RUNX3) inTranscription Regulation of Natural Cytotoxicity Receptor 1(NCR1/NKp46), an Activating Natural Killer (NK) CellReceptor*□S

Received for publication, September 22, 2011, and in revised form, January 5, 2012 Published, JBC Papers in Press, January 17, 2012, DOI 10.1074/jbc.M111.306936

C. Benjamin Lai and Dixie L. Mager1

From the Terry Fox Laboratory, British Columbia Cancer Agency and Department of Medical Genetics, University of BritishColumbia, Vancouver, British Columbia V5Z1L3, Canada

Background: The expression of the activating receptor NKp46/NCR1 is highly restricted to NK cells.Results: RUNX proteins bind identified cis-regulatory element and affect NCR1 expression.Conclusion: NCR1 is regulated by two key proximal cis-regulatory elements and by RUNX factors.Significance:Mapping regulatory components of NCR1 will contribute to a deeper understanding of NK biology.

Natural cytotoxicity receptor 1 (NCR1), also known asNKp46, is a natural killer (NK) lymphocyte-activating receptor.It is involved in major aspects of NK immune function andshows a high degree of lineage specificity in blood and bonemarrow. The nature of its NK-restricted expression is not wellunderstood. In this study, we confirm that human NCR1 NK-specific expression is achieved at themRNA level.We found twokey cis-regulatory elements in the immediate vicinity upstreamof the gene. One element acts as an essential promoter, whereasthe other acts as a tissue-dependent enhancer/repressor. Thislatter regulatory element contains a runt related-transcriptionfactor (RUNX) recognition motif that preferentially bindsRUNX3. Interferingwith RUNXproteins using a dominant neg-ative form results in decreased Ncr1 expression. RUNX3 over-expression had the opposite effect. These findings shed light onthe role of RUNX3 in the control of an important NK-activatingreceptor.

Natural killer (NK)2 cells are large granular lymphocytes thathave antiviral, anticancer, and immunoregulatory functions (1).Recent findings suggest that they possess memory reminiscentof adaptive T and B cells (2). However, they have long beenconsidered as part of the innate branch due to their reliance ongerm-line encoded receptors. The large number of NK recep-

tors can be categorized into inhibiting, typified by NKG2A, theLy49, and the killer immunoglobulin-like receptors, and acti-vating, including NKG2D and the natural cytotoxicity recep-tors (3).NKp46 is an activating receptor encoded by natural cytotox-

icity receptor 1 (NCR1). It is a type 1 transmembrane proteincontaining two immunoglobulin-like domains, initially identi-fied via a redirected killing assay screen (4). Monoclonal anti-body-mediated cross-linking of NKp46 induces calcium mobi-lization, cytolytic activity, aswell as interferon� (IFNG) release.Only one natural ligand has been discovered for NKp46 so far:the viral hemagglutinin (5). However, NCR1/NKp46 is postu-lated to have unknown tumor ligands that can participate intumor immunoediting (6). The emerging role ofNKp46 is high-lighted in studies on mice deficient for Ncr1. These knock-outmice are unable to fight influenza infection, unable to control aspecific experimental lymphoma model, and are more pro-tected from development of type 1 diabetes (7–9).NCR1 is unique amongNK surface receptors. Unlike some of

the other receptors, NCR1 is highly conserved in mammals(10–12). Perhaps most interestingly, NCR1 and NCR3 are theonly NK receptors that show relatively high expression speci-ficity (13). Recent studies indicate that NKp46 also marksmouse gutRORC�IL22� innate immune cells andhuman ton-sil CD127�RORC� lymphoid tissue inducer-like cells (14, 15),calling into question the specificity of the receptor. However, atleast in the bone marrow and blood, NKp46 acts as a bona fideNK marker (16).The expression pattern ofNCR1 alludes to specific transcrip-

tional control. Thus, studying its regulation provides an oppor-tunity to identify important transcription factors of the naturalkiller lineage. Furthermore, theNCR1 promoter is on the vergeof becoming a widely used and efficacious tool to study thebiology of conventional NK cells. Already, one study has used itto ablate NK cells in vivo, whereas another used it to create anNK-specific Stat5-deficient mouse (16, 17). However, themechanism behind its precise control has so far gone largelyunaddressed. Here we focus on the proximal upstream regionof the human NCR1 gene. We identify two cis-regulatory ele-

* This work was supported by a grant from the Canadian Institutes of HealthResearch with core support provided by the British Columbia CancerAgency.

□S This article contains supplemental Table S1 and Figs. S1–S3.1 To whom correspondence should be addressed: Terry Fox Laboratory, BC

Cancer Agency, 675 West 10th Ave., Vancouver, British Columbia V5Z1L3,Canada. Tel.: 604-675-8139; Fax: 604-877-0712; E-mail: dmager@bccrc.ca.

2 The abbreviations used are: NK, natural killer; NCR, natural cytotoxicityreceptor; IFNG, interferon �; RUNX, runt-related transcription factor;hRUNX, human RUNX; RORC, retinoic acid related orphan receptor c;PBMC, peripheral blood mononuclear cells; APC, allophycocyanin; PE, phy-coerythrin; ACTB, � actin; ETS, E-twenty six family; dn-RUNX, dominantnegative RUNX; H3K4me3, histone 3 lysine 4 trimethylation; H3K27me3,histone 3 lysine 27 trimethylation; �c, common � chain; Bis-Tris, 2-(bis-(2-hydroxyethyl)amino)-2-(hydroxymethyl)propane-1,3-diol; NKP, NKprogenitor.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 10, pp. 7324 –7334, March 2, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

7324 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

ments in this region and describe a role of runt-related tran-scription factor (RUNX) proteins, especially RUNX3, in regu-lating NCR1 expression.

EXPERIMENTAL PROCEDURES

PBMCFlowCytometry—Peripheral bloodwas obtained fromhealthy donors, and peripheral blood mononuclear cells(PBMC) were isolated by density centrifugation on FicollPLUS (StemCell Technologies). PBMC were then washed withPBS containing 5% human serum (SigmaH4522), and Fc recep-tors were blocked using human Fc receptor blocking reagent(Miltenyi). Next, the cells were stained using monoclonal anti-bodies: CD335/NKp46-APC (Miltenyi 130-092-609), CD3-FITC (BD Biosciences 555332), CD56-PE (StemCell Technolo-gies 10526), CD14-FITC (StemCell Technologies 10406),CD15-FITC (BD Biosciences 555401), CD19-PE (BeckmanCoulter IM1285U), and CD33-PE (BD Biosciences 347787).Isotype control antibodies were: IgG1-APC (BD Biosciences555751), IgG1-FITC (StemCell Technologies 10310), IgG1-PE(StemCell Technologies 10311), IgG2b-FITC (BD Biosciences555742), IgG2b-PE (BD Biosciences 555743), and IgM-FITC(BD Biosciences 555583). Lastly, propidium iodide was addedto 5 �g/ml. Four-color analysis was carried out usingFACSCalibur.Cell Culture—NK92, a humanNK cell line (18), was cultured

in minimum essential medium Eagle’s with Earle’s salts andnonessential amino acids, supplemented with 12.5% (v/v) fetalbovine serum, 12.5% (v/v) horse serum, 2 mM L-glutamine, 100�M 2-mercaptoethanol, 100 units/ml IL-2 (PeproTech). KY-2and LNK (both mouse NK cell lines) were cultured in RPMI1640medium supplemented with 10% (v/v) fetal bovine serum,2 mM L-glutamine, and 50 �M 2-mercaptoethanol. LNK andKY-2 used 1000 units/ml and 200 units/ml of IL-2, respectively.U2OS (human osteosarcoma), K562 (human erythroleukemia),LoVo (human colorectal adenocarcinoma), HEK-293 (humanembryonic kidney), and NIH-3T3 (mouse embryonic fibro-blast) cells were cultured in Dulbecco’s modified Eagle’smedium supplemented with 10% (v/v) fetal bovine serum (fetalcalf serum forNIH-3T3). IM-9 (humanB-lymphoblastoid), Jur-kat (human T-lymphocyte), THP-1 (human acute monocyticleukemia), and HL-60 (human acute promyelocytic leukemia)cell lines were cultured in RPMI 1640 medium supplementedwith 10% (v/v) fetal bovine serum.MouseNKcellswere isolatedfrom spleen of Runx3-deficient (19) and wild type mice by neg-ative selection (R&D MagCollect mouse NK cell isolation kit,MAGM210) and cultured for 7 days in RPMI 1640 mediumcontaining 10% (v/v) fetal calf serum, 2 mM L-glutamine, 1%sodium pyruvate, 1% nonessential amino acids, 50 �M

2-mercaptoethanol, and 1000 units/ml IL2 (BiologicalIndustries, Beit-Haemek, Israel, 30-T209B). All cultureswere supplemented with 100 units/ml penicillin, 100units/ml streptomycin.RT-PCRandReal-timePCR—Total RNA fromcellswere col-

lected using RNeasy mini kit and QIAshredder homogenizer(Qiagen). RNA samples were treated with Turbo DNase(Ambion) before cDNA conversion using SuperScript III andrandomprimers (Invitrogen). Primers used for PCRare listed insupplemental Table S1. Real-time PCRwas done using the 7500

Fast real-time PCR system (Applied Biosystems). Relativeexpression was determined using the 2���CTmethod. Thresh-old cycles were normalized to Actb.Generation of Luciferase Reporter Constructs—Promoter

fragments were generated by PCR using forward primers withKpnI site inserted at the 5� end and reverse primers with NheIsite inserted at the 5� end. PCR products were digested withKpnI/NheI (New England Biolabs) and cloned into pLG4.10(pGL4B) firefly luciferase promoter vector (Promega). RUNXmutations were introduced into the 5�-270 construct using theQuikChange Lightning site-directed mutagenesis kit (Strat-agene). For all primer sequences, see supplemental Table S1.Transient Transfection and Luciferase Assays—NK92 and

U2OS cells were transiently transfected with LipofectamineLTXwith PLUS reagent (Invitrogen) according tomanufactur-er’s instructions. Briefly, 1.0 � 105 NK92 cells and 5.0 � 105U2OS cells were seeded into 500 �l of growthmediumwithoutpenicillin/streptomycin in 24-well plates for 24 h at 37 °C. Foreach well, 100 �l of LTX transfection medium was prepared: 2�l of LTX reagent, 1 �g of vector, 0.1 �g of pRL-TK, 1 �l ofPLUS, topped up with medium without serum. The mix wasincubated for 30min prior to addition to wells. Cells were incu-bated at 37 °C for 24 h before assaying.Luciferase activity was assayed using the Dual-Luciferase

reporter assay system (Promega) according to the manufactur-er’s instructions. Firefly luciferase activity was normalized rel-ative to Renilla luciferase activity for each transfection andcalculated as -fold increase over pGL4.10-BASIC (pGL4B).Western Blot—Nuclear extraction was performed as de-

scribed before (20). Protein concentration was determinedusing the NanoDrop ND-1000 (Thermo Scientific). NuPAGENovex 4–12% Bis-Tris gels (Invitrogen) were used to resolvethe protein sample in MOPS SDS running buffer (Invitrogen).Proteins were transferred onto an Immobilon-P PVDF mem-brane (Millipore) in transfer buffer (25 mM Tris, 192 mM gly-cine, 10%methanol, 0.1% SDS). Themembranewas blocked for1 h with blocking solution: 5% (w/v) skim milk reconstitutedwith TBST buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween 20).Primary antibody was then added at 1:3000 anti-RUNX3(Abcam ab11905) and 1:3000 anti-ACTB (Sigma A2066) over-night at 4 °C with constant agitation. The membrane waswashed five times with TBST buffer at room temperature with5-min intervals. Goat anti-rabbit (SigmaA0545) at 1:10,000wasdiluted in TBST buffer with 0.5% (w/v) of skimmilk. Themem-brane was incubated in this secondary antibody solution for 1 hat room temperature under constant agitation. Washes werecarried out as above. Protein was detected using SuperSignal(Thermo Scientific) and Kodak BioMax MR film.EMSA—Wild type and mutated double-stranded probes

containing the putative RUNX binding sites were generated byannealing oligomers (oligomers listed in supplemental TableS1). Nuclear extraction, probe labeling, and the gel shift assaywere performed as described before (20). Seven �g of proteinwas used per binding reaction (protein concentration wasdetermined using the NanoDrop ND-1000 (Thermo Scien-tific)). For supershift experiments, 2.5 �l of anti-RUNX3(Abcam ab11905) and 2.5 �g of anti-RUNX1 (Abcam ab23980)was used.

RUNX3 Regulates NCR1/NKp46

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7325

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Chromatin Immunoprecipitation—ChIP assay was per-formed as described previously (21) with the following modifi-cations. Formaldehyde cross-linking was done for 15 min atroom temperature for cell lines and 10min for mouse NK cells.Sonication was done using 30-s on, 30-s off cycles (25 cycles forNK92, 30 cycles for U2OS and IM-9, 20 cycles for mouse NKcells). Preclearing and pulldown utilized protein A/G beads(Thermo Scientific). Immunoprecipitation was accomplishedwith 5 �l or 5 �g of anti-RUNX3 (Abcam ab11905), anti-RUNX1 (Abcam ab23980), anti-histone 3 lysine 4 trimethyla-tion (Millipore CS200580), anti-histone 3 lysine 27 trimethyla-tion (Abcam ab6002), and normal rabbit IgG (Millipore).Retrovirus Transduction and Fluorescence-assisted Cell

Sorting—Control MIGR1, dominant negative RUNX (dn-RUNX) on MIGR1 backbone (HA-NLS-Runt), and humanRUNX3 (hRUNX3) cDNA on MIY backbone (MIY-hRUNX3)have been described previously (22, 23). These vectors weretransiently transfected into Plat-E (24) using TurboFect in vitroreagent (Fermentas) according to the manufacturer’s instruc-tions. Forty-eight hours later, viral supernatant was collectedand used immediately or stored in�80 °C. The growthmediumof KY-2 cells was switched with viral supernatant plus 4 �g/mlPolybrene (Sigma) and 200 units/ml IL-2 (PeproTech). For theMIY-hRUNX3 experiments, the viral supernatant wasswitched back to growth medium after 24 h. For the dn-RUNXexperiments, KY-2 cells were subjected to a second round ofinfection as above after 24 h.Another 24 hpassed for the secondround before the KY-2 culture was switched back to growthmedium. Two or 4 days after the switch, fluorescence-assistedcell sorting was used to isolate GFP� or YFP� cells. Cells wereallowed to recover from sorting stress for 24 h in growthmedium before RNA collection.

RESULTS

NKp46/NCR1 Expression Specificity Stems from TranscriptLevel—NKp46/NCR1 is widely accepted as a bona fide NKmarker at least in blood. There is good evidence for NKp46specificity at the cell surface in both human and mouse (4, 16).Indeed, in healthy human PBMC, almost all NKp46� cells alsoexpress CD56, but not T, B, or myeloid lineage markers CD3,CD19, CD14, CD15, and CD33 (Fig. 1A). However, few havechecked whether the expression pattern is reflected at the tran-scriptional level in human.We addressed this issue by perform-ing semiquantitative RT-PCRon a panel of human cell lines andsurveying publicly available microarray data. In accordancewith a previous study (25), out of a panel of human cell lines(NK, T, B, monocyte, promyeloblast, erythroid, osteosarcoma,and embryonic kidney), only the NK cell line shows NCR1expression (Fig. 1B). In primary tissue, as shown by publishedmicroarray data, NCR1 transcripts are also confined to the NKlineage (Fig. 1C). These results suggest that the NK-specificexpression of NKp46 is transcriptionally controlled and thatthe cell line, NK92, is a good NK model to further study themechanism of its regulation.Human NCR1 Promoter Contains Essential and Enhancing

Region—Walzer et al. (16) were able to drive NK-specificexpression using just 400 bp of noncoding sequence upstreamof the human NCR1 gene, suggesting that all the important

FIGURE 1. NCR1 transcript is NK-specific. A, phenotypic analysis ofhealthy human PBMC. Freshly isolated PBMC were stained for NKp46 andother indicated lineage markers. Dot plots show representative data fromthree independent experiments. B, RT-PCR detection of NCR1 in humancell lines with ACTB as internal control. Total RNA from indicated cell lineswas used for reverse transcriptase reactions. C, microarray of NCR1 expres-sion. Data were accessed through BioGPS (51). Human data are fromGeneAtlas U133A probe set 207860_at (52). Mouse data are from Gene-Atlas MOE430 probe set 1422089_at (53). Only relevant hematopoietic celltypes are shown. Pro, progenitor; DC, dendritic cell; HSC, hematopoieticstem cell.

RUNX3 Regulates NCR1/NKp46

7326 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

cis-regulatory elements are immediately upstream of the geneitself. In agreement with this, the level of genomic conservationupstream of the start codon of humanNCR1 is limited to�300bp (supplemental Fig. S1). To further pinpoint the crucial cis-regulatory elements, we cloned�396 to�1 relative to theATGinto pGL4B (designated FL; Fig. 2). We also made truncationmutants designated with the suffix 5� or 3� (depending on thedirection of truncation) followed by the size of the remainingpromoter. These constructs were transiently transfected intoNK92 cells, and luciferase assays were conducted. The 5�-95construct, corresponding to the region immediately upstreamof the ATG, shows appreciable promoter activity (�4-foldabove background). This fragment acts as an essential pro-moter because its removal, as seen in the 3� truncations, com-pletely abrogates activity. In addition, we observe a significantgain of promoter strength between 5�-200 and 5�-270 con-structs (from 6- to 13-fold above background). Thus, thereappears to exist an enhancer between �270 and �200. Thedifferences between 5�-95 and 5�-200, or between FL and5�-270, were not statistically significant. We conclude thatthere are no major cis-regulatory sequences between �95 to�200 and �270 to �396. These human constructs showed asimilar trend in the mouse NK cell line, KY-2, suggesting thatthe promoter is not species-specific (supplemental Fig. S2).NCR1 Promoter Contains RUNX Sites, and RUNX Members

Are Expressed in NK Cells—To identify the transcription fac-tors that regulate the NCR1 promoter, we scanned the pro-

moter sequence for transcription factor binding motifs usingthe Transcription Element Search System (TESS). Curatedresults containing only mammalian hematopoietic transcrip-tion factors are shown in Fig. 3A. To narrow down the list, wefocused on the essential and enhancing cis-regulatory regionsand took two approaches. Firstly, we carried out a semiquanti-tative RT-PCR screen to check the expression of the predictedtranscription factors in NK versus other cell types. None of thepredicted factors had an expression pattern as specific asNCR1(Fig. 3B). However, we noticed that RUNX3/AML2 was con-fined mostly to the lymphoid lineage and was relatively high inNK92 cells. RUNX3 is closely related to two other runt familymembers, RUNX1 and RUNX2. We focused on RUNX1 andRUNX3, quantitatively confirming their expression by real-time PCR. The results mirrored the semiquantitative RT-PCRresults for the most part (Fig. 3C). Human cell lines expressvarying amounts of RUNX1 when compared with NK92.RUNX3was highest in NK92 and IM-9, whereas other cell linesexpressed undetectable levels or levels more than 5-fold lesswhen compared with NK92.Using semiquantitative RT-PCR, we also checked Runx1 and

Runx3mRNA inmouse NK cell lines, LNK and KY-2, as well asnon-NK cell lines, NIH-3T3 and Ms1. In the mouse NK celllines, Ncr1 and Runx3 are detectable, whereas in the non-NKcell lines, only Runx1 is detectable (supplemental Fig. S3).These human andmouse results agree with primary tissue datafrompublicly availablemicroarray studies (Fig. 3D, supplemen-tal Fig. S3).Next, we aligned �400 bp upstream ofNCR1 from 10 mam-

malian species using European Bioinformatics Institute (EBI)ClustalW2 and checked whether the binding motifs identifiedby TESS were conserved (Fig. 3A, supplemental Fig. S1). Thisalignment revealed that only two RUNX sites and one ETS sitewere highly conserved. The enhancing region harbors oneRUNXsite thatmatches theRUNXconsensus (ACCACA). Theessential region contains an imperfect binding site (ACCACT)and an ETS site. Both the expression data and the bindingmotifconservation at NCR1 promoter point toward RUNX proteinsas possible regulators of NCR1.Human andMouse NK Cells Use Distal Promoter of RUNX3—

The presence of RUNX3 transcripts does not guarantee func-tional RUNX3 protein. In both human and mouse, expressionof RUNX3 is regulated by two promoters (26, 27). AlthoughRunx3mRNAcan be detected in bothmouseCD8� andCD4�T cells, only the CD8� population expresses the distal tran-script isoform and detectable levels of RUNX3 protein (28, 29).In mouse NK cells, the distal form of Runx3 transcript isexpressed starting at the CD122� NK1.1�, NKP stage. RUNXprotein has also been found in mouse NK lysates using a pan-RUNX antibody (30).The transcription factor screen in Fig. 3, B and C, utilized

primers that did not differentiate between proximal and distalRUNX3. To resolve this issue, we performed RT-PCR usingpromoter-specific primers. In NK92 cells, the distal transcriptwas preferentially expressed, and the proximal transcript wasbarely detectable (Fig. 4B). By Western blotting, RUNX3 pro-tein can be seen in NK92 nuclear extracts (Fig. 4C). Therefore,

FIGURE 2. Luciferase reporter assay of NCR1 promoter in NK92. A diagramof the promoter and first three exons of NCR1 is shown at the top. 5�-UTR andcoding regions are shown as thin and thick gray boxes, respectively. The blackarrow depicts the TSS. Promoter fragments truncated from the 5� and 3� endswere cloned into pGL4B and transiently transfected into NK92. Firefly lucifer-ase activity was assayed normalized to co-transfected Renilla luciferase activ-ity. Promoter strength is calculated as -fold above pGL4B empty vector. Datashown represent means with standard deviations of more than three exper-iments. Tests of significance were carried out using analysis of variance fol-lowed by Tukey’s post test (***, p � 0.001).

RUNX3 Regulates NCR1/NKp46

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7327

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

humanNKcells express the distal formofRUNX3at themRNAand protein levels.RUNX Binding Motif Mutations Decrease NCR1 Promoter

Strength—Theessential andenhancing regionscontainawell con-served RUNX binding site each.We hypothesized that the recog-nition motifs were important to NCR1 promoter activity. Muta-

tions were introduced to these motifs in our pGL4B constructcontaining the promoter from�1 to�270. Luciferase assayswerethen performed in NK92 cells. Destroying the RUNX binding sitein the enhancing region results in a drop of promoter strengthfrom 13- to 4-fold above background (Fig. 5). When the samemutation was introduced into the motif in the essential region,

FIGURE 3. Identification of RUNX sites in NCR1 promoter. A, TESS search for transcription factor binding sites in the NCR1 promoter. Potential binding motifsare indicated in brackets. Only mammalian hematopoietic related factors are shown. Lowercase and uppercase denote intergenic and genic sequences,respectively. Sequences in bold indicate the enhancing and essential regions. TBP, TATA-binding protein; NFAT, nuclear factor of activated T-cells; CREB,cAMP-response element-binding protein; C/EBPa, CCAAT-enhancer-binding protein �. B, RT-PCR detection of transcription factors in human cell lines. TotalRNA from indicated cell lines was used for reverse transcriptase reactions. Primers as indicated under “Experimental Procedures” were used to detect varioustranscription factors and ACTB. C, real-time PCR detection of RUNX1 and RUNX3. Transcript levels of the two RUNX members were assayed and normalized toACTB. mRNA levels in the NK92 cell line were set to one. Data shown represent means with standard deviations of three experiments. D, microarray of RUNX3expression. Data accessed through BioGPS (51) from GeneAtlas U133A probe set 207860_at (52). Only relevant hematopoietic cell types are shown.

RUNX3 Regulates NCR1/NKp46

7328 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

promoter strength decreased to 3-fold above background. How-ever, we cannot rule out a contribution from the ETS binding sitebecause the mutation overlaps both the putative RUNX site andthe putative ETS site. The results indicate that these motifs arerequired for optimal expression from theNCR1 promoter.

RUNX3 and RUNX1 Bind Promoter in Vitro—All membersof the runt family bind the conserved motif, 5�-ACCRCA-3�.We wondered whether either RUNX1 or RUNX3 can indeedbind the predicted motifs in the NCR1 promoter. To test thispossibility, electrophoretic mobility shift assays (EMSA) wereperformed. We tested the two RUNX sites separately. Probesspanning the putative RUNX binding site in the enhancer wereshifted by NK92 nuclear extract (Fig. 6). Two bands are clearlyvisible, labeled I and II. The bands were completely abolishedwith the introduction of unlabeled wild type competing oligo-mers at 200-fold excess. If unlabeled competing oligomers witha mutated RUNX site were used instead, both band I and bandII were still visible, although weaker. The decrease in signalstrength is likely the result of nonspecific binding of mutantprobes at molar excess. We confirmed that the identities of thebinding proteins were RUNX3 and RUNX1 by supershiftexperiments. When anti-RUNX3 or anti-RUNX1 was incu-bated with NK92 nuclear proteins beforehand, a supershiftedband III was observed. Band II, whose signal strengthdecreased, most likely contains the RUNX3/RUNX1-DNAcomplexes. The identity of band I is currently unknown.Probes spanning the predicted RUNX binding site in the

essential region were also shifted by NK92 nuclear extract (Fig.6). Again, multiple bands are visible, labeled IV and V. Thebands completely disappear with competition from wild typebut notmutant unlabeled oligomers at 200-fold excess. Neitheranti-RUNX3 nor anti-RUNX1 antibodies supershifted thesebands. Thus, the nuclear proteins binding the essential regionand forming bands IV and V are neither RUNX1 nor RUNX3.Side-by-side comparison of shift patterns between the essential

FIGURE 6. RUNX forms protein-DNA complexes with NCR1 promoter ele-ments in vitro. EMSA was performed using probes containing RUNX motifs inthe enhancer (lanes 2– 6) and essential (lanes 7–11) region. A side-by-sidecomparison of band shift patterns between the two regions is shown in lanes12 and 13. As control, lane 1 contains only �-32P-labeled WT enhancer andessential probe. NK92 nuclear extract (NK92 nuc. extract) was incubated withlabeled WT probe at 4 °C for 20 min (lanes 2 and 7). For competition assays,unlabeled competitors were incubated with NK92 nuclear extract at 4 °C for20 min before the addition of labeled WT probe. Competitors were used at200-fold excess (lanes 3, 4, 8, and 9). Supershift assays were carried out usinganti-RUNX3 (lanes 5 and 10) or anti-RUNX1 (lanes 6 and 11) antibodies. I, II, IV,and V represent band shifts of DNA-protein interaction. III represents bandsupershift of DNA-protein-antibody interaction.

FIGURE 4. NK cells express distal RUNX3. A, diagram of RUNX3 gene struc-ture at the 5� end. Thin and thick gray boxes represent 5�-UTR and codingregions, respectively. Arrows indicate primers used to detect RUNX3 mRNAisoforms: distal forward, proximal forward, and common reverse primers.B, RT-PCR detection of RUNX3 isoforms in human NK cell lines. Total RNA fromNK92 was used for reverse transcriptase reactions. C, Western blot analysis ofRUNX3 protein in NK92. Immunoblotting was performed on nuclear extracts.Arrows in the upper panel correspond to the positions of RUNX3 protein (usinganti-human RUNX3 antibody), and the lower panel shows the position ofACTB protein (loading control).

FIGURE 5. RUNX motif mutations decrease NCR1 promoter strength.Mutations shown at the top were introduced into a promoter fragment from�1 to �270 in pGL4B backbone. The vector was transiently transfected intoNK92, and luciferase activity was measured using a Dual-Luciferase system.Firefly luciferase activity was normalized to co-transfected Renilla luciferaseand calculated as -fold above the pGL4B empty vector. Data shown representmeans with standard deviations of three experiments.

RUNX3 Regulates NCR1/NKp46

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7329

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

and enhancer regions show that the band positions are not thesame, further hinting that RUNX proteins are not binding theessential region. The most likely candidates remaining are ETSfamily members, based on our motif prediction and reporterassay results. Taken together, these results suggest that RUNX1and RUNX3 both bind the enhancer region in the NCR1promoter.RUNX3andRUNX1Bind Promoter in Vivo—In vitro binding

of transcription factors toDNA is not always reflective of in vivostates due to the variability of chromatin context.We thereforeexamined whether RUNX1 and RUNX3 were enriched at thehuman NCR1 promoter specifically in NK cells by using chro-matin immunoprecipitation (ChIP). As a negative control, wechose an intergenic region 8 kb downstream of NCR1. Thisregion is not known to contain any regulatory sequences. As apositive control, we analyzed the promoter ofCD122.We chosethis because in mouse NK cells, the Cd122 promoter was pre-viously shown to bind RUNX protein using a pan-RUNX anti-body (30). We performed the ChIP assay using anti-humanRUNX3- and anti-humanRUNX1-specific antibodies. InNK92cells, the negative control region did not show significantenrichment of either transcription factor (Fig. 7A). The CD122promoter shows significant enrichment of RUNX1 (7-fold) andRUNX3 (11-fold) over isotype background. At the NCR1 pro-moter, we also detected significant enrichment of RUNX1(5-fold) and RUNX3 (16-fold).TodeterminewhetherRUNXbinding to theNCR1promoter

is NK-specific, ChIP was performed in other cell lines. Wechose IM-9 and U2OS. When compared with NK92, IM-9expresses equivalent levels of RUNX3 transcripts, but 10-foldless RUNX1. On the other hand, U2OS expresses equivalentlevels of RUNX1 transcripts, but 25-fold less RUNX3 (Fig. 3C).Neither of these cells showed enrichment for RUNX1 orRUNX3 at NCR1 promoter (Fig. 7A). Thus, RUNX transcrip-tion factors bind NCR1 promoter in an NK-specific manner.In a Runx3-deficient mouse, flow cytometric analysis

revealed that NKp46 is expressed at a level comparable withwild type.3 We wondered whether Runx1 can compensate forRunx3 in regulatingNcr1 expression. To test this possibility, weperformed ChIP on NK cells isolated from Runx3 knock-outand wild type mice. In wild type NK cells, we could not detectRunx1 enrichment at the Ncr1 promoter when compared withisotype levels. However in Runx3-deficient NK cells, RUNX1 isenriched at the same location by about 3-fold (Fig. 7B). Theseresults indicate that in mouse NK cells, RUNX1 does not bindNcr1 promoter normally but takes the place of RUNX3 whenthe latter is absent.Dominant Negative RUNX Interferes with NCR1 Trans-

cription—To test the effects of all RUNX proteins on NCR1expression, we used mouse dn-RUNX (30). dn-RUNX consistsof the DNA binding Runt domain of RUNX transcription fac-tors, without the transactivation domain. Because all RUNXmembers bind the same motif, dn-RUNX acts as a pan-RUNXcompetitive inhibitor. We retrovirally introduced dn-RUNX into KY-2 and isolated successful transductants at two

time points, days 4 and 6 after transduction. The isolation wasachieved by fluorescence-activated cell sorting (FACS) usingthe GFP marker on the vector. Transcript levels of variousgenes were assayed by real-time PCR.18S RNA, which acts as a negative control, did not change

significantly throughout the time points (Fig. 8). As positivecontrols, we looked at Ifng, Prf1 (perforin 1), and Cd43 (sialo-phorin). Interferon � and perforin have been shown to be reg-ulated by Runx3 in mouse CD4� and CD8� T cells (31–34).3 D. Levanon, personal communication.

FIGURE 7. RUNX binds NCR1 promoter in vivo. ChIP was performed in NK92,IM-9, and U2OS (A) and mouse wild type and Runx3-deficient NK cells (B) usinganti-human RUNX3, anti-human/mouse RUNX1, and IgG isotype control. Pre-cipitated DNA was assayed by real-time PCR using primers specific for thehuman NCR1 promoter, an intergenic region 8 kb downstream of humanNCR1, the human CD122 promoter, and the mouse Ncr1 promoter. Enrich-ment is calculated as -fold over IgG control. Data shown represent means withstandard deviations of three experiments.

RUNX3 Regulates NCR1/NKp46

7330 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Additionally, Ohno et al. (30) showed that interferon � andCD43 are perturbed in dn-RUNX-expressing NK cells. Whencompared with empty vector-transduced KY-2 cells, dn-RUNX-transduced cells show decreased expression of all posi-tive control genes to �50% for Ifng and Prf1 and 73% for CD43on day 4. On day 6, the down-regulation of these genes wasmore pronounced, ranging from 17 to 55% expression whencompared with empty vector control. Ncr1 was similarlydecreased in expression at both timepoints (44 and 22%ondays4 and 6when comparedwith empty vector, respectively). Theseresults suggest that the RUNXproteins are required for optimaltranscription of Ncr1, differentiation markers, and effectorgenes in mouse NK cells.RUNX3 Overexpression Increases NCR1 Expression—To

more directly examine the role of RUNX3 in regulation ofNCR1, we decided to overexpress RUNX3 in NK cells. Wetransduced human RUNX3 (hRUNX3) cDNA into KY-2 cellsand selected using the vector YFP by FACS. Human RUNX3expression in mouse KY-2 was verified by real-time PCR (Fig.9A). Overexpression of hRUNX3 did not lead to an increase inendogenous mouse Runx3, nor the negative controls, 18S RNA

and �-2 microglobulin (Fig. 9B). Interestingly, Prf1mRNA lev-els did not change. Additionally, although Ifng levels seemed toincrease mildly, the change was not statistically significant.BothCd43 andNcr1mRNA increased almost 4-foldwhen com-pared with empty vector control. These results imply thatRUNX3 specifically contributes to Cd43 and Ncr1 expression,whereas other RUNX family members may be more importantin regulating Prf1 and Ifng.Promoter Behaves Differently in Non-NK Cells—We won-

dered whether the essential and enhancer elements of theNCR1 promoter were tissue-specific. Thus, we ran luciferaseassays using the 5� truncation constructs in the U2OS cell line.In these cells, the constructs behaved differently when com-pared with the NK92 cells (Fig. 10A). The essential region byitself still confers high promoter activity, but the regionbetween �200 and �270 actually down-regulates promoterstrength. This trend is also observed in HeLa cells (data notshown), suggesting that the effects are not an artifact of theU2OS cell line. Thus, the essential region acts primarily as apan-tissue promoter. On the other hand, the �200 to �270region acts as an enhancer or suppressor, depending on thecellular context (henceforth termed the switch region).Furthermore, we investigated the chromatin context of the

promoter in NK92 and IM-9 cells. To this end, we performedChIP using antibodies toward histone modifications histone 3lysine 4 trimethylation (H3K4me3) and histone 3 lysine 27trimethylation (H3K27me3). H3K4me3 is a chromatin markstrongly linked to the promoters of actively transcribing genes,whereas the H3K27me3 mark is considered repressive andlinked to silent loci (35). H3K4me3 was found to be highlyenriched at the NCR1 promoter only in NK92 cells (Fig. 10B).Conversely, at the same location, IM-9 cells display preferentialenrichment of H3K27me3. Thus, the chromatin configurationis favorable to expression in NK cells and closed in non-NKcells.

DISCUSSION

Tissue-restricted expression is not an uncommon phenom-enon.What makesNCR1 unique is the small number of knownNK-specific genes. Even more remarkable is the fact that thespecificity can arise from a short proximal promoter of �270bp. The luciferase assay results further show the lineage-limit-ing sequence to be confined to a 70-bp region.Within the switch region, we found a runt bindingmotif. The

binding motif is the same for all members of the runt family:RUNX1, RUNX2, and RUNX3. RUNX1 is a crucial transcrip-tion factor in the hematopoietic system. Its deficiency results inblocked hematopoiesis during development, and conditionalmutants have abnormalities in lymphoid as well as myeloid lin-eages (36). The transcription factor is also known to regulatemultiple hematopoietic genes (37). RUNX2 is primarilyinvolved in bone formation and not so much in hematopoiesis(37). Finally, RUNX3 is a key player in T cell development andfunction. Runx3 knock-out models indicate that the protein isrequired for cytotoxic T lymphocyte specification, down-regu-lating CD4, and up-regulating CD8 (38, 39). RUNX3 is re-ex-pressed when naive CD4 T cells differentiate into TH1 but notTH2 cells (31). In CD8 T cells and TH1 cells, RUNX3 controls

FIGURE 8. dn-RUNX decreases Ncr1 expression. Gene expression was meas-ured by real-time PCR. Actb is the endogenous control. Expression levels indn-RUNX transductants were normalized to levels in empty vector controltransductants. Data shown represent means with standard deviations ofthree experiments.

FIGURE 9. RUNX3 overexpression enhances Ncr1 expression. Gene expres-sion was measured by real-time PCR with Actb as the endogenous control.A, expression of human RUNX3 levels in MIY-hRUNX3 transductants withrespect to endogenous mouse Runx3 (mRunx3, set to 1). B, expression of var-ious genes in these transductants normalized to levels in empty vector con-trol transductants. Data shown represent means with standard deviations ofthree experiments.

RUNX3 Regulates NCR1/NKp46

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7331

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

multiple effector function genes including granzyme, inter-feron �, and perforin. Indeed, the current paradigm indicatesboth RUNX1 and RUNX3 to be important players in T celldifferentiation (37, 40).In NK cells, the importance of the RUNX proteins is only

beginning to emerge. The human killer immunoglobulin-likereceptor (KIR) genes are known to have RUNX binding sites,and variants with mutated RUNX sites are not expressed (41).The cofactor for the RUNX proteins, core binding factor �(Cbf�), is required for propermouse NK cell development (42).Furthermore, Runx proteins regulate genes such as Cd122 andLy49, and Runx3 undergoes a dramatic up-regulation duringNK differentiation; it is low in hematopoietic stem cells andpeaks at the immature NK stage (30, 42). Certainly, when com-paring different hematopoietic cell types, RUNX3 seems to beconsistently overexpressed in NK cells in human andmouse. Inour cell lines, only IM-9 matched the high level of RUNX3expression in NK cells. It is known that naive B cells usuallyexpress RUNX1, but switch to RUNX3 when transformedby Epstein-Barr virus (43). This could explain why IM-9, anEpstein-Barr virus-transformed B lymphoblastic cell line, hasRUNX3 mRNA levels as high as NK92 cells. We link RUNX1and RUNX3 directly to an important determinant of NK func-tion by showing that these transcription factors bind a RUNXsite in the endogenous NCR1 promoter. A pan-RUNX domi-nant negative protein disrupts optimal NCR1 expression,whereas RUNX3 overexpression has the opposite effect.Although both RUNX1 and RUNX3 can bind the enhancerregion, the higher level of RUNX3 expression in NK cells mayresult in preferred binding of RUNX3 at the NCR1 promoter.We hypothesize that RUNX1may act as a redundant factor andbind the promoter if RUNX3 is disrupted. This redundancy isnot surprising if one takes into account the relative importanceof the NCR1 receptor in host immune defense.Our dn-RUNX results also show a down-regulation of Ifng

and Prf1, indicating a link between the RUNX proteins and NKeffector function. In contrast, Ohno et al. (30) showed that dn-RUNX up-regulated Ifng. This discrepancy may be due to sev-eral fundamental differences in methodology. Ohno et al. (30)used NK1.1�CD3� cells from spleen of dn-RUNX transgenicmice to test intracellular IFNG protein levels. We used a dn-RUNX-transduced mouse NK cell line and tested transcript

levels by real-time PCR. It is also possible that RUNX affectsIfng differently at transcriptional and translational levels.Already, the limited amount of evidence available for

RUNX3 in NK, CD8� T, and TH1 cells implicates the tran-scription factor as an important mediator of cell-mediatedimmunity. Of course, this notion suggests that RUNX3 alone isnot enough to direct NK specificity, instead merely potentiatestranscription in cytotoxic and TH1 lymphocytes. In fact, nosingle transcription factor has been shown to conclusively act asa “master regulator” of the NK program so far. Such a factorwould be required for the expression of the NK program andrepression of non-NK genes. Another possibility is that multiplefactors that are not lineage-specific act in a combinatorial way torestrict expression of NK genes such as NCR1. RUNX3 could besuch a factor, and the search is still on to identify others.To date, no studies have investigated theNKcompartment in

the existing complete Runx3 knock-out mouse models (44).With the development of theNcr1-cremouse, it is now possibleto delete genes specifically in the NK lineage. Although Ncr1 isunder the control of Runx3, it should still be possible to selec-tively delete the Runx3 locus. Studies that examine the down-stream targets of RUNX3 could lead to new perspectives in NKcell biology.The pathway leading toRUNX3 expression is also of interest.

Park et al. (45) recently showed that RUNX3 is induced by com-mon � chain (�c) receptor signaling during CD8 T specifica-tion. The process is dependent on STAT5 or STAT6 as �c-re-lated cytokine stimulation of thymocytes from Stat5/6 doubleknock-out mouse did not induce Runx3 expression. Major �creceptors are also known to play important roles in NK devel-opment and stimulation (46). Downstream of the receptors,Stat5 deficiency in NKwas recently shown to lead to a blockageat the Lin-CD122� NKP stage (17). Finally, RUNX proteinspositively regulate interleukin-2 receptor � (IL2R�)/CD122 inNK cells (30). The link between STAT5/6 and RUNX3 in NKcells still needs to be experimentally established. However, theemerging picture is one where the IL-15 receptor, which firstappears in the NKP stage, activates the appropriate STATmembers including STAT5 in cells committed to the NK line-age. This axis may lead to induction of transcription factorsincluding RUNX3 to turn on NK-related genes such as NCR1.RUNX3, in turn, acts in a positive feedback loop to further

FIGURE 10. NCR1 promoter in non-NK cells. A, 5� truncation promoter constructs as in Fig. 2 were transiently transfected into U2OS cells. Firefly luciferaseactivity was assayed normalized to co-transfected Renilla luciferase activity. Promoter strength is calculated as -fold above pGL4B empty vector. Data shownrepresent means with standard deviations of more than three experiments. Tests of significance were carried out using analysis of variance followed by Tukey’spost test (***, p � 0.001). B, ChIP was performed in NK92 and IM-9 cells using anti-human H3K4me3, anti-human H3K27me3, and IgG isotype control.Precipitated DNA was assayed by real-time PCR using primers specific for the NCR1 promoter. Enrichment is calculated as -fold over IgG control. Data shownrepresent means with standard deviations of three experiments.

RUNX3 Regulates NCR1/NKp46

7332 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

up-regulate IL2R�/CD122, a subunit of the IL-15 receptor. Infact, surface NKp46 can be forcibly induced in a small propor-tion of human CD8� T cells (�5%), when these cells are cul-tured ex vivo for 12 days with IL-15 (47). This suggests that theaxis plays an important, although by itself not sufficient, role inthe NK program.Aside from the runt binding motif, we also noticed an ETS

binding motif that can contribute toNCR1 transcriptional reg-ulation. The ETS family transcription factors have many mem-bers, and a few in particular are known to participate in NKdevelopment and function (48). Furthermore, we previouslyshowed that GA-binding protein (GABP), another member ofthe family, is required for optimal althoughnot absolute expres-sion of the mouse Nkg2d long isoform (49). The ETS motif intheNCR1 promoter lies in the essential region, which does notseem to be responsible for tissue specificity. Nevertheless,mutations to the binding site affect the activity of the promoter.Although an imperfect RUNX binding site was identified rightnext to the ETS site, we could not detect binding of RUNX1 norRUNX3 to this site in vitro. Thus, these RUNXproteins seem tobe solely involved in enhancing promoter activity in the NKlineage, whereas ETS proteinsmay be involved in setting up thebasal activity.In non-NK cells, we observed that the essential region by

itself has appreciable promoter activity. However, this activityis suppressed by the switch region. The flip of function in thiscis-regulatory region is intriguing as it suggests that NK speci-ficity requires a dual mode of control: context-dependentenhancer and repressor. The identity of the suppressing tran-scription factor remains amystery. The RUNXproteins do pos-sess repressor activity depending on bound co-repressors orhistone deacetylases (50). However, they are not bound to theNCR1 promoter in non-NK cells and thus are unlikely to beinvolved in repression of NCR1 expression. Histone modifica-tions act as an additional level of tissue-specific control becausewe found the enrichment of permissive and repressive chroma-tin marks in NK and non-NK cell lines, respectively.An understanding of NCR1 regulation bears clinical as well

as research potential. Because mouse models show a strongrelationship between the receptor and diseases such as influ-enza and diabetes, one clinical goal is to efficaciously managethe expression of the receptor to benefit disease outcomes.Such studies also provide insight into the understanding of theNK program, which noticeably lags behind our understandingof T and B lymphocyte programs. As the usage of the NCR1promoter for transgenic studies gains traction, rational manip-ulation of the promoter will provide greater flexibility in theusage of this great tool.

Acknowledgments—We thank Dr. Ditsa Levanon and VardaNegreanu for sharing Runx3 knock-out mouse NK cells as well asvaluable discussion and advice. The RUNX dominant negative andRUNX3 overexpression retrovirus constructs were kind gifts from Dr.Takehito Sato and Dr. Aly Karsan. We are grateful for technical helpand discussions from Dr. Ping Xiang, Samuel Gusscott, ChristopherJenkins, and Rebecca Cullum. We also thank the Terry Fox Labora-tory FACS facility staff for cell sorting.

REFERENCES1. Vivier, E., Tomasello, E., Baratin, M., Walzer, T., and Ugolini, S. (2008)

Functions of natural killer cells. Nat. Immunol. 9, 503–5102. Paust, S., and von Andrian, U. H. (2011) Natural killer cell memory. Nat.

Immunol. 12, 500–5083. Colucci, F., Di Santo, J. P., and Leibson, P. J. (2002) Natural killer cell

activation in mice and men: different triggers for similar weapons? Nat.Immunol. 3, 807–813

4. Sivori, S., Vitale, M., Morelli, L., Sanseverino, L., Augugliaro, R., Bottino,C., Moretta, L., and Moretta, A. (1997) p46, a novel natural killer cell-specific surface molecule that mediates cell activation. J. Exp. Med. 186,1129–1136

5. Mandelboim,O., Lieberman,N., Lev,M., Paul, L., Arnon, T. I., Bushkin, Y.,Davis, D. M., Strominger, J. L., Yewdell, J. W., and Porgador, A. (2001)Recognition of hemagglutinins on virus-infected cells by NKp46 activateslysis by human NK cells. Nature 409, 1055–1060

6. Elboim, M., Gazit, R., Gur, C., Ghadially, H., Betser-Cohen, G., andMandel-boim, O. (2010) Tumor immunoediting by NKp46. J. Immunol. 184,5637–5644

7. Gazit, R., Gruda, R., Elboim,M., Arnon, T. I., Katz,G., Achdout,H.,Hanna, J.,Qimron, U., Landau, G., Greenbaum, E., Zakay-Rones, Z., Porgador, A., andMandelboim,O. (2006) Lethal influenza infection in the absence of the natu-ral killer cell receptor gene Ncr1.Nat. Immunol. 7, 517–523

8. Halfteck, G. G., Elboim,M., Gur, C., Achdout, H., Ghadially, H., andMan-delboim, O. (2009) Enhanced in vivo growth of lymphoma tumors in theabsence of the NK-activating receptor NKp46/NCR1. J. Immunol. 182,2221–2230

9. Gur, C., Porgador, A., Elboim, M., Gazit, R., Mizrahi, S., Stern-Ginossar,N., Achdout, H., Ghadially, H., Dor, Y., Nir, T., Doviner, V., Hershkovitz,O., Mendelson, M., Naparstek, Y., and Mandelboim, O. (2010) The acti-vating receptor NKp46 is essential for the development of type 1 diabetes.Nat. Immunol. 11, 121–128

10. Kelley, J., Walter, L., and Trowsdale, J. (2005) Comparative genomics ofnatural killer cell receptor gene clusters. Plos. Genet. 1, 129–139

11. Storset, A. K., Kulberg, S., Berg, I., Boysen, P., Hope, J. C., and Dissen, E.(2004) NKp46 defines a subset of bovine leukocytes with natural killer cellcharacteristics. Eur. J. Immunol. 34, 669–676

12. Jozaki, K., Shinkai, H., Morozumi, T., Tanaka-Matsuda, M., Eguchi-Ogawa, T., Wada, Y., and Uenishi, H. (2010) Cloning, expression, andpolymorphisms of natural killer cell receptor NCR1 in pigs. Anim. Bio-technol. 21, 156–163

13. Bottino, C., Biassoni, R., Millo, R., Moretta, L., andMoretta, A. (2000) Thehuman natural cytotoxicity receptors (NCR) that induce HLA class I-in-dependent NK cell triggering. Hum. Immunol. 61, 1–6

14. Satoh-Takayama, N., Vosshenrich, C. A., Lesjean-Pottier, S., Sawa, S.,Lochner,M., Rattis, F.,Mention, J. J., Thiam, K., Cerf-Bensussan, N.,Man-delboim, O., Eberl, G., and Di Santo, J. P. (2008) Microbial flora drivesinterleukin 22 production in intestinal NKp46� cells that provide innatemucosal immune defense. Immunity 29, 958–970

15. Crellin, N. K., Trifari, S., Kaplan, C. D., Cupedo, T., and Spits, H. (2010)Human NKp44�IL-22� cells and LTi-like cells constitute a stableRORC� lineage distinct from conventional natural killer cells. J. Exp.Med. 207, 281–290

16. Walzer, T., Bléry, M., Chaix, J., Fuseri, N., Chasson, L., Robbins, S. H.,Jaeger, S., André, P., Gauthier, L., Daniel, L., Chemin, K., Morel, Y., Dalod,M., Imbert, J., Pierres, M., Moretta, A., Romagné, F., and Vivier, E. (2007)Identification, activation, and selective in vivo ablation of mouse NK cellsvia NKp46. Proc. Natl. Acad. Sci. U.S.A. 104, 3384–3389

17. Eckelhart, E., Warsch, W., Zebedin, E., Simma, O., Stoiber, D., Kolbe, T.,Rülicke, T., Mueller, M., Casanova, E., and Sexl, V. (2011) A novel Ncr1-Cre mouse reveals the essential role of STAT5 for NK cell survival anddevelopment. Blood 117, 1565–1573

18. Gong, J. H., Maki, G., and Klingemann, H. G. (1994) Characterization of ahuman cell line (NK-92) with phenotypical and functional characteristicsof activated natural killer cells. Leukemia 8, 652–658

19. Levanon, D., Bettoun, D., Harris-Cerruti, C., Woolf, E., Negreanu, V.,Eilam, R., Bernstein, Y., Goldenberg, D., Xiao, C., Fliegauf, M., Kremer, E.,

RUNX3 Regulates NCR1/NKp46

MARCH 2, 2012 • VOLUME 287 • NUMBER 10 JOURNAL OF BIOLOGICAL CHEMISTRY 7333

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

Otto, F., Brenner, O., Lev-Tov, A., and Groner, Y. (2002) The Runx3 tran-scription factor regulates development and survival of TrkC dorsal rootganglia neurons. EMBO J. 21, 3454–3463

20. Maksakova, I. A., and Mager, D. L. (2005) Transcriptional regulation ofearly transposon elements, an active family of mouse long terminal repeatretrotransposons. J. Virol. 79, 13865–13874

21. Wederell, E. D., Bilenky, M., Cullum, R., Thiessen, N., Dagpinar, M., De-laney, A., Varhol, R., Zhao, Y., Zeng, T., Bernier, B., Ingham,M., Hirst, M.,Robertson, G., Marra, M. A., Jones, S., and Hoodless, P. A. (2008) Globalanalysis of in vivo Foxa2 binding sites in mouse adult liver using massivelyparallel sequencing. Nucleic Acids Res. 36, 4549–4564

22. Hayashi, K., Natsume,W.,Watanabe, T., Abe, N., Iwai, N., Okada, H., Ito,Y., Asano, M., Iwakura, Y., Habu, S., Takahama, Y., and Satake, M. (2000)Diminution of the AML1 transcription factor function causes differentialeffects on the fates of CD4 and CD8 single-positive T cells. J. Immunol.165, 6816–6824

23. Fu, Y., Chang, A. C., Fournier, M., Chang, L., Niessen, K., and Karsan, A.(2011) RUNX3 maintains the mesenchymal phenotype after terminationof the Notch signal. J. Biol. Chem. 286, 11803–11813

24. Morita, S., Kojima, T., and Kitamura, T. (2000) Plat-E: an efficient andstable system for transient packaging of retroviruses. Gene Ther. 7,1063–1066

25. Pessino, A., Sivori, S., Bottino, C., Malaspina, A., Morelli, L., Moretta, L.,Biassoni, R., and Moretta, A. (1998) Molecular cloning of NKp46: a novelmember of the immunoglobulin superfamily involved in triggering of nat-ural cytotoxicity. J. Exp. Med. 188, 953–960

26. Rini, D., and Calabi, F. (2001) Identification and comparative analysis of asecond runx3 promoter. Gene 273, 13–22

27. Bangsow, C., Rubins, N., Glusman, G., Bernstein, Y., Negreanu, V., Gold-enberg, D., Lotem, J., Ben-Asher, E., Lancet, D., Levanon, D., and Groner,Y. (2001) The RUNX3 gene: sequence, structure, and regulated expres-sion. Gene 279, 221–232

28. Sato, T., Ohno, S., Hayashi, T., Sato, C., Kohu, K., Satake, M., and Habu, S.(2005) Dual functions of Runx proteins for reactivating CD8 and silencingCD4 at the commitment process into CD8 thymocytes. Immunity 22,317–328

29. Egawa, T., Tillman, R. E., Naoe, Y., Taniuchi, I., and Littman, D. R. (2007)The role of the Runx transcription factors in thymocyte differentiationand in homeostasis of naive T cells. J. Exp. Med. 204, 1945–1957

30. Ohno, S., Sato, T., Kohu, K., Takeda, K., Okumura, K., Satake, M., andHabu, S. (2008) Runx proteins are involved in regulation of CD122, Ly49family, and IFN-� expression duringNK cell differentiation. Int. Immunol.20, 71–79

31. Djuretic, I. M., Levanon, D., Negreanu, V., Groner, Y., Rao, A., and Ansel,K. M. (2007) Transcription factors T-bet and Runx3 cooperate to activateIfng and silence Il4 in T helper type 1 cells. Nat. Immunol. 8, 145–153

32. Wang, L.,Wildt, K. F., Castro, E., Xiong, Y., Feigenbaum, L., Tessarollo, L.,and Bosselut, R. (2008) The zinc finger transcription factor Zbtb7b re-presses CD8 lineage gene expression in peripheral CD4� T cells. Immu-nity 29, 876–887

33. Yagi, R., Junttila, I. S., Wei, G., Urban, J. F., Jr, Zhao, K., Paul, W. E., andZhu, J. (2010) The transcription factor GATA3 actively represses RUNX3protein-regulated production of interferon-�. Immunity 32, 507–517

34. Cruz-Guilloty, F., Pipkin, M. E., Djuretic, I. M., Levanon, D., Lotem, J.,Lichtenheld, M. G., Groner, Y., and Rao, A. (2009) Runx3 and T-boxproteins cooperate to establish the transcriptional program of effectorCTLs. J. Exp. Med. 206, 51–59

35. Barski, A., Cuddapah, S., Cui, K., Roh, T. Y., Schones, D. E.,Wang, Z.,Wei,G., Chepelev, I., and Zhao, K. (2007) High-resolution profiling of histonemethylations in the human genome. Cell 129, 823–837

36. Growney, J. D., Shigematsu,H., Li, Z., Lee, B.H., Adelsperger, J., Rowan, R.,Curley, D. P., Kutok, J. L., Akashi, K., Williams, I. R., Speck, N. A., and

Gilliland, D. G. (2005) Loss of Runx1 perturbs adult hematopoiesis and isassociated with a myeloproliferative phenotype. Blood 106, 494–504

37. Cohen, M. M., Jr. (2009) Perspectives on RUNX genes: an update. Am. J.Med. Genet. A. 149A, 2629–2646

38. Taniuchi, I., Osato, M., Egawa, T., Sunshine, M. J., Bae, S. C., Komori, T.,Ito, Y., and Littman, D. R. (2002) Differential requirements for Runx pro-teins in CD4 repression and epigenetic silencing during T lymphocytedevelopment. Cell 111, 621–633

39. Woolf, E., Xiao, C., Fainaru, O., Lotem, J., Rosen, D., Negreanu, V., Bern-stein, Y., Goldenberg, D., Brenner, O., Berke, G., Levanon, D., andGroner,Y. (2003) Runx3 and Runx1 are required for CD8 T cell developmentduring thymopoiesis. Proc. Natl. Acad. Sci. U.S.A. 100, 7731–7736

40. Collins, A., Littman, D. R., and Taniuchi, I. (2009) RUNX proteins intranscription factor networks that regulate T cell lineage choice.Nat. Rev.Immunol. 9, 106–115

41. Vilches, C., Gardiner, C. M., and Parham, P. (2000) Gene structure andpromoter variation of expressed and nonexpressed variants of theKIR2DL5 gene. J. Immunol. 165, 6416–6421

42. Guo, Y., Maillard, I., Chakraborti, S., Rothenberg, E. V., and Speck, N. A.(2008) Core binding factors are necessary for natural killer cell develop-ment and cooperate with Notch signaling during T cell specification.Blood 112, 480–492

43. Spender, L. C., Cornish, G. H., Sullivan, A., and Farrell, P. J. (2002) Expres-sion of transcription factor AML-2 (RUNX3, CBF(�)-3) is induced byEpstein-Barr virus EBNA-2 and correlates with the B cell activation phe-notype. J. Virol. 76, 4919–4927

44. Levanon, D., and Groner, Y. (2009) Runx3-deficient mouse strains circa2008: resemblance and dissimilarity. Blood Cells Mol. Dis. 43, 1–5

45. Park, J. H., Adoro, S., Guinter, T., Erman, B., Alag, A. S., Catalfamo, M.,Kimura, M. Y., Cui, Y., Lucas, P. J., Gress, R. E., Kubo, M., Hennighausen,L., Feigenbaum, L., and Singer, A. (2010) Signaling by intrathymic cyto-kines, not T cell antigen receptors, specifies CD8 lineage choice and pro-motes the differentiation of cytotoxic lineage T cells. Nat. Immunol. 11,257–264

46. Williams, N. S., Klem, J., Puzanov, I. J., Sivakumar, P. V., Schatzle, J. D.,Bennett, M., and Kumar, V. (1998) Natural killer cell differentiation: in-sights from knockout and transgenic mouse models and in vitro systems.Immunol. Rev. 165, 47–61

47. Correia, M. P., Costa, A. V., Uhrberg, M., Cardoso, E. M., and Arosa, F. A.(2011) IL-15 induces CD8� T cells to acquire functional NK receptorscapable of modulating cytotoxicity and cytokine secretion. Immunobiol-ogy 216, 604–612

48. Hesslein, D. G., and Lanier, L. L. (2011) Transcriptional control of naturalkiller cell development and function. Adv. Immunol. 109, 45–85

49. Lai, C. B., Zhang, Y., Rogers, S. L., andMager, D. L. (2009) Creation of thetwo isoforms of rodent NKG2Dwas driven by a B1 retrotransposon inser-tion. Nucleic Acids Res. 37, 3032–3043

50. Durst, K. L., and Hiebert, S. W. (2004) Role of RUNX family members intranscriptional repression and gene silencing. Oncogene 23, 4220–4224

51. Wu, C., Orozco, C., Boyer, J., Leglise, M., Goodale, J., Batalov, S., Hodge,C. L., Haase, J., Janes, J., Huss, J. W., 3rd, and Su, A. I. (2009) BioGPS: anextensible and customizable portal for querying and organizing gene an-notation resources. Genome Biol. 10, R130

52. Su, A. I.,Wiltshire, T., Batalov, S., Lapp, H., Ching, K. A., Block, D., Zhang,J., Soden, R., Hayakawa, M., Kreiman, G., Cooke, M. P., Walker, J. R., andHogenesch, J. B. (2004) A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl. Acad. Sci. U.S.A. 101, 6062–6067

53. Lattin, J. E., Schroder, K., Su, A. I., Walker, J. R., Zhang, J., Wiltshire, T.,Saijo, K., Glass, C. K., Hume, D. A., Kellie, S., and Sweet, M. J. (2008)Expression analysis of G Protein-Coupled Receptors in mouse macro-phages. Immunome Res. 4, 5

RUNX3 Regulates NCR1/NKp46

7334 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 10 • MARCH 2, 2012

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from

C. Benjamin Lai and Dixie L. Mager(NK) Cell Receptor

), an Activating Natural KillerNCR1/NKp46of Natural Cytotoxicity Receptor 1 () in Transcription RegulationRUNX3Role of Runt-related Transcription Factor 3 (

doi: 10.1074/jbc.M111.306936 originally published online January 17, 20122012, 287:7324-7334.J. Biol. Chem. 

  10.1074/jbc.M111.306936Access the most updated version of this article at doi:

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

  http://www.jbc.org/content/suppl/2012/01/17/M111.306936.DC1

  http://www.jbc.org/content/287/10/7324.full.html#ref-list-1

This article cites 53 references, 19 of which can be accessed free at

by guest on Novem

ber 17, 2020http://w

ww

.jbc.org/D

ownloaded from